Granulocyte colony-stimulating factor (GCSF) is one of the hematopoietic growth factors with multifunctional activities. As a glycoprotein, GCSF plays important regulatory functions in the processes of maturation, proliferation, differentiation, and functional activation of granulocyte precursors and mature granulocytes in the bone marrow. It is able to augment white blood cell production when bone marrow dysfunction exists. Recombinant DNA technology has made it possible to clone the genes responsible for GCSF and to develop pharmaceutical products to treat a number of human hematopoietic conditions and disorders such as neutropenia and hematopoietic stem cell transplantation.
Human GCSF (hGCSF) protein has a molecular mass of 19.6 kDa and exerts its biological functions through binding to the human GCSF receptor (hGCSFR), a single transmembrane protein with a large extracellular region that consists of an immunoglobulin-like (Ig-like) domain, a cytokine receptor homology (CRH) domain, and three fibronectin type III domains. Binding of GCSF to the extracellular Ig-like and CRH domains of the receptor triggers receptor homodimerization with a 2:2 stoichiometry of hGCSF/hGCSFR (Tamada T, et al. 2006 Homodimeric cross-over structure of the human GCSF receptor signaling complex. PNAS 103:3135-3140). The dimerization results in activation of intracellular Janus tyrosine kinase-signal transducers and activators of transcription (Jak-Stat) type signaling cascade. The signaling transfer of hematopoietic factor receptors from extracellular region to intracellular cascades has been suggested to be via conformational changes of the receptor dimer in TM domains. It has been demonstrated that the dimeric erythropoietin (EPO) receptor can be activated by mutations at the TM domain in the absence of the natural ligand EPO, a hematopoietic growth factor regulating red blood cell production (Lu X, et al. 2006 Active conformation of the erythropoietin receptor: Random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains. JBC 281:7002-7011). Patients with mutations in TM domain of hGCSFR have experienced chronic neutrophilia due to the receptor constitutive activation (Plo I, et al. 2009 An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia. JEM 206:1701-1707).
Development of bioengineered EPO and GCSF targeted binding sites of the natural ligands have been successful, while development of a protein-based thrombopoietin (TPO) drug has failed due to drug-related increase in antibody production against endogenous TPO. A peptide-based TPO mimic molecule, romiplostim, recently received FDA approval, although the risk of drug-related antibody development is to be monitored in longer term use. Discovery efforts to find small molecules targeting the same site have not been fruitful. Eltrombopag is the first FDA approved small molecule drug in the hematopoietic growth factor field for the treatment of thrombocytopenia. Different from romiplostim that competes with endogenous TPO for the same binding site of TPO receptor, eltrombopag activates TPO receptor most likely by interacting with the TM domain and, as a result, its activity is additive to that of endogenous TPO. Eltrombopag showed a unique species-specific TPO receptor activation that requires histidine-499, and partially activated a mouse GCSF receptor where a cysteine residue in the TM domain was mutated to histidine (Erickson-Miller C, et al. 2004 Species specificity and receptor domain interaction of small molecule TPO receptor agonists. ASH 2004 San Diego, poster). Several classes of compounds have been reported to activate the TPO receptor. For example, WO 2004/033433, WO 2007/062078, WO 2006/047344, WO 01/89457, WO 2009/103218, U.S. Pat. No. 7,026,334, WO 2005/014561, WO 2005/007651, WO 2006/033005, WO 2007/004038, WO 2007/036769, and WO 2007/054783.
A small molecule that selectively activates mouse GCSF receptor has been identified. (Tian S-S, et al. 1998 A small, nonpeptidyl mimics of granulocyte-colony-stimulating factor. Science 281:257-259). This molecule is reported to not directly compete with GCSF, although it seems to bind to the extracellular region of mouse GCSF receptor. (Doyle M L, et al. 2003 Selective binding and oligomerization of the murine GCSF receptor by a low molecular weight, nonpeptidyl ligand. JBC 278:9426-9434). A class of small molecules have been reported to activate the both mouse and human GCSF receptors and the site of action is not suggested (Kusano K, et al. 2004 A potential therapeutic role for small nonpeptidyl compounds that mimic human GCSF. Blood 103:836-842; Tokizawa M, et al. 2004 Imidazole derivatives of their salts. U.S. Pat. No. 6,737,434).